Potential utility of urinary chemokine CCL2 to creatinine ratio in prognosis of 5‐year graft failure and mortality post 1‐year protocol biopsy in kidney transplant recipients

Abstract Background Chemokines (chemotactic cytokines) are small proteins which are engaged in many pathophysiological processes, including inflammation and homeostasis. In recent years, application of chemokines in transplant medicine was intensively studied. The aim of this study was to determine the utility of urinary chemokines CCL2 (C‐C motif ligand 2) and CXCL10 (C‐X‐C motif chemokine ligand 10) in prognosis of 5‐year graft failure and mortality post 1‐year protocol biopsy in renal transplant recipients. Methods Forty patients who had a protocol biopsy 1 year after renal transplantation were included. Concentrations of CCL2 and CXCL10 in urine with reference to urine creatinine were measured. All patients were under the supervision of one transplant center. Long‐term outcomes within 5 years after 1‐year posttransplant biopsy were analyzed. Results Urinary CCL2:Cr at the time of biopsy was significantly increased in patients who died or had graft failure. CCL2:Cr was proven to be a significant predictor of 5‐year graft failure and mortality (odds ratio [OR]: 1.09, 95% confidence interval [CI]: 1.02–1.19, p = .02; OR: 1.08, 95% CI: 1.02–1.16, p = .04; respectively). Conclusion Chemokines are easily detected by current methods. In the era of personalized medicine, urinary CCL2:Cr can be considered as a factor providing complementary information regarding risk of graft failure or increased mortality.


| INTRODUCTION
Chemokines (chemotactic cytokines) are heparinbinding proteins that have sizes between 5 and 20 kDa. They are engaged in many pathophysiological processes, including inflammation and homeostasis. 1 Chemokines can bind glycosaminoglycans located on the surface of endothelial cells and in the extracellular matrix, including heparan sulfate. 2 Moreover, chemokines interact with several receptors and most of the chemokines' receptors can be recognized by different ligands. Those receptors belong to seven transmembrane G-protein coupled receptors, which are localized predominantly on the surface of leukocytes. 3 Glycosaminoglycanschemokines-receptors interactions are intensively studied. 4 Chemokines are able to bind heparan sulfate which is a glycosaminoglycan widely expressed on cell surfaces and in the extracellular matrix, but also it can be derived from heparin-anticoagulation drug. 5 Anti-inflammatory effect of heparin is under investigation. 6 Chemokines are engaged in mediating graft injury during different stages of transplantation, which reflects inflammatory/innate and donor-specific/acquired immune responses. The production of chemokines is noted early during the reperfusion of graft, during episodes of acute rejection and during the development of vasculopathy and fibrosis. 7,8 One of the first discovered chemokine is CCL2 (C-C motif ligand 2), also called monocyte chemoattractant protein-1 (MCP-1). 9 CCL2 plays an important role in many inflammatory diseases and is involved in the development of renal diseases. CCL2 is found mainly in endothelial cells, fibroblasts, and mononuclear cells. The gene for CCL2 is situated on the q-arm of chromosome 17. 10 Expression of CCL2 is regulated by various molecules. Interleukin 1, tumor necrosis factor alpha, and interferon gamma are the main inducers of CCL2 expression. The inhibition of CCL2 expression is caused by anti-inflammatory agents as glucocorticosteroids, estrogen, and retinoic acid. [11][12][13] CCL2 binds mainly to CC chemokine receptor 2 (CCR2) which triggers cells such as monocytes and other immune cells that stimulate inflammation. It is a major factor driving leukocyte infiltration. CCL2 contributes to fibrosis by influencing macrophages. 14 The role of CCL2 was described in numerous kidney diseases, such as IgA nephropathy, membranous nephropathy, glomerulosclerosis, autosomal dominant polycystic kidney disease, lupus nephritis, GBM-induced nephritis, ANCA-associated renal vasculitis, and diabetic nephropathy. 15 CCL2 can be easily detected in urine. Therefore, its use as a biomarker of kidney disease has been broadly analyzed. 16 Moreover, it was shown that patients after kidney transplantation (KTx) diagnosed with acute or chronic rejection have higher concentrations of CCL2 in urine and serum. 17 During the rejection process, CCL2 produced in response to inflammatory stimulation, is actively involved in the recruitment of macrophages into the graft. The damaging impact of macrophages in allograft rejection is still being explored. 18 Similarly to CCL2, chemokine CXCL10 (C-X-C motif chemokine ligand 10) has a role in the pathogenesis of kidney diseases such as lupus nephritis or kidney allograft dysfunction. 19 CXCL10 is also classified as interferon gamma-induced protein 10 (IP-10). CXCL10 secretion is stimulated by interferon gamma, which implies its role in the development of inflammation and fibrosis. 20 It binds principally to the CXCR3 receptor which is located mainly on activated T lymphocytes, and also on B-cells and natural killer cells, which results in their mobilization into allograft and intensification of immune reaction. 21 CXL10 has been proved to be associated with T-cell-mediated rejection and antibodymediated rejection. 22,23 Currently, a multicenter randomized controlled trial protocol of urine CXCL10 monitoring strategy in kidney transplant recipients is being performed. 24 KTx is the treatment of choice for patients with endstage kidney disease. However, there is a worldwide shortage of organ donors and new patients are added to the waiting list for renal transplant every day. 25 Currently, the median graft survival for deceased donor transplants is estimated to be 11.7 years. 26 Many risk factors of kidney graft failure are determined: those linked to the donor, to surgical procedures and to recipients. 27,28 Antibody-mediated rejection is recognized as the major cause of renal allograft failure. 29 Early identification of the risk factors of graft failure or mortality could influence clinical decisions and be crucial in maintaining stable graft function and in prolonging patient survival. 30 The aim of the study was to determine the utility of urinary chemokines CCL2 and CXCL10 in prognosis of 5-year graft failure and mortality post 1-year protocol biopsy in renal transplant recipients.

| Study population
This is a noninvasive study based on previous research, which its original aim was to analyze urinary concentrations of chemokines CCL2 and CXCL10 as the biomarkers of harmful processes in renal allograft. 31 T A B L E 1 Characteristics of study population. Age at biopsy, years; mean (SD) Primarily, the single-center study consisted of 40 patients, who underwent a protocol biopsy 1 year after a receiving kidney transplant from a brain-dead deceased donor between the years 2015 and 2017. The ultrasoundguided biopsy procedure was done when the patients did not have any signs of infection and did not demonstrate any complaints. All the specimens were assessed according to the Banff classification by one renal pathologist. 32 All patients were Caucasians and received a maintenance immunosuppression which included tacrolimus, mycophenolate mofetil, and steroids.
According to the biopsy results, four groups of patients were identified: patients with interstitial fibrosis and tubular atrophy (IFTA) grade II or III (N = 16), BK virus (BKV) nephropathy (N = 4), patients with mild inflammatory lesions fulfilling the criteria for mild rejection processes or borderline lesions (N = 11), and kidney recipients with IF/TA grade I or without any pathological changes (N = 15, constituted control group).
In this investigation, the patients' long-term outcomes 5 years after the 1-year posttransplant biopsy were assessed. During the follow-up, all patients were under the supervision of our transplant center. The data were collected 5 years postbiopsy within 3 months range when patients did not present no signs of infection, nor exacerbation of chronic disease. Graft failure was defined as the need for permanent dialysis or retransplantation. Mortality from all causes was considered, including patients with failed graft.

| Chemokine analysis
The analysis of urinary concentrations of chemokines was done according to protocol described previously. 31 To correct for dilution of the urine, the concentrations of CCL2 and CXCL10 were expressed with reference to urinary creatinine. 33 At the time of biopsy, urine samples were collected and stored for further analysis. An enzyme-linked immunosorbent assay (ELISA) was used for a quantitative measurement of chemokines (R&D Systems). For each patient, samples were tested twice, and the mean value was used for analysis.

| Statistical analysis
Mean values with standard deviation or medians with quartiles 1 and 3 (Q1-Q3) were used to represent categorical data, while percentages were used to represent continuous data. The Fisher exact test was used for categorical variables were compared using the Fisher exact test. Continuous variables were compared using, the two-sample t test or Mann-Whitney test. The Shapiro-Wilk test was used to assess normality of distribution.
The predictive value for graft failure and patient death was analyzed using a univariate logistic regression model. Due to the relatively small sample size, multivariate regression analysis was not performed. Odds ratios (OR) with 95% confidence intervals (CI) were calculated.
p Value < .05 was considered statistically significant.

| RESULTS
Patients' characteristics are depicted in Table 1. As it was described in a previous paper, the groups did not differ in age, sex, immunologic features (first transplantation, human leukocyte antigen [HLA] mismatch), and blood concentration of tacrolimus at the time of biopsy. 31 Outcomes of the patients 5 years after the 1-year posttransplant biopsy were analyzed. Regarding the immunosuppressive regimen, all patients with functioning graft still received triple immunosuppression consisting of tacrolimus, mycophenolate mofetil, and steroids. During the 5-year follow-up, five patients experienced graft failure and seven patients died (three patients died with functioning graft). Graft failure was caused by chronic antibody-mediated rejection (N = 2), chronic allograft dysfunction of unknown etiology (N = 2), and recurrent urinary tract infections (N = 1). The causes of death were cardiovascular events (N = 5) or malignancy (N = 2). Table 2 shows a comparison of patients who had graft failure or died to other patients. Alive patients with functioning graft had significantly lower serum creatinine concentration at the time of biopsy and significantly higher estimated glomerular filtration rate (eGFR) Chronic Kidney Disease Epidemiology Collaboration (CKD EPI). The urinary chemokine CCL2-to-creatinine ratio (CCL2:Cr) at the time of biopsy was significantly increased in patients who had graft failure or died. In contrast, the urinary chemokine CXCL10-to-creatinine ratio (CXCL10:Cr) ratio did not differ between groups. There was significant difference regarding IFTA stage between patients with graft failure and alive patients with functioning graft.
Prognostic values of serum creatinine, eGFR CKD EPI, urinary CCL2:Cr ratio, and IFTA stage 1-year posttransplant for graft failure or mortality are depicted in Table 3. Serum creatinine, eGFR CKD EPI, and urinary CCL2:Cr at the time of biopsy proved to be significant predictors of graft failure or mortality within 5 years. IFTA stage was identified as a significant predictor of graft failure. Age at biopsy, years; mean (SD) 48 Abbreviations: CCL2, C-C motif ligand 2; CKD EPI, chronic kidney disease epidemiology collaboration; CXCL10, C-X-C motif chemokine ligand 10; eGFR, estimated glomerular filtration rate; HLA, human leukocyte antigen; IFTA, interstitial fibrosis and tubular atrophy; SD, standard deviation. a p Values compared with alive patients with functioning graft. GNIEWKIEWICZ ET AL.

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Early transplant outcomes have improved over time as a result of better surgical methods, postoperative management, and modern immunosuppressives (especially calcineurin inhibitors). Long-term results, on the other hand, have not changed to the same level, which remains challenging for transplant specialists. 34 There is an emerging need for the ability to early detect renal allografts with a high risk of failure. Specific biomarkers with practical diagnostic and prognostic value are demanded. 35 Urine is the most common specimen for renal diseases because it includes molecules and cell components derived precisely from glomerular filtration of plasma, excretion of kidney tubules, and secretion of the urinary tract that demonstrate pathophysiological and metabolic processes in human beings at a specific point in time. Urine samples can be obtained recurrently, in a fully noninvasive way, in reasonably large volumes. 36,37 In the study, patients with IFTA stage II or III diagnosed in biopsy 1 year after transplantation had significantly higher incidence of 5-year graft failure or 5-year mortality compared with patients without any pathological changes detected by biopsy. Moreover, IFTA was identified to be a predictor of graft failure. Similar results were shown in the literature. 38,39 IFTA is a feature of progressive chronic allograft dysfunction, which is a multifactorial entity and one of the main causes of long-term graft failure. Typically, it does not give any symptoms and develops 1 year after transplantation. It results in steady decline in kidney function. 40 The prompt diagnosis of this process is important, since failure to do so results in permanent changes and eventually to graft failure. 41 The molecular processes engaged in chronic allograft dysfunction happen very early. However, they do not have clinical demonstration, and it is not possible to identify them by common techniques. Diagnosis of IFTA requires a renal biopsy, which is an invasive procedure. 42 In relation to the results, it is broadly demonstrated in available papers that renal function at 1-year posttransplant is associated with long-term outcomes. Both serum creatinine and eGFR CKD EPI are significantly associated with graft failure and mortality. 30,43,44 Nevertheless, serum creatine is affected by multiple factors including age, gender, race, body mass, diet, some of which are eliminated by glomerular filtration rate estimated equitation. 45 Glomerular filtration rate measurement by iothalamate or other direct methods is still considered the gold standard for assessing renal function, but it would be impractical to apply this technique in routine clinical practice. 46 Previous papers have shown that urinary concentration of CCL2 is elevated in patients with advanced IFTA or graft rejection. 31,47 Our data indicates that urinary CCL2:Cr 1-year posttransplant is associated with 5-year graft failure and mortality. Some publications display related findings. Increased concentration of CCL2 in kidney transplant recipients with poor outcomes may be explained by its contribution to repair after kidney injury. 48,49 CCL2 is involved in pathogenic processes during all stages of transplantation, including vasculopathy and fibrosis. 7 CCL2 is a potent chemoattractant that regulates migration and infiltration of macrophages. Macrophage infiltration identified in 1-year posttransplant protocol biopsies was associated with subclinical alloimmune inflammation, injury of tubules, and progression of fibrosis. 50 In the study, mortality, could be explained by previous occurrences of graft failure (four patients out of seven died with previous graft failure). The presence of a failed renal graft after cessation of immunosuppressive treatment can lead to a state of chronic inflammation, which reduces patient survival. 51,52 There was no statistical difference regarding urinary CXCL10:Cr between patients who had graft failure or died and patients with stable graft function. It is intriguing as CXCL10 is determined as an independent Abbreviations: CCL2, C-C motif ligand 2; CI, confidence interval; CKD-EPI, Chronic Kidney Disease Epidemiology Collaboration; eGFR, estimated glomerular filtration rate; IFTA, interstitial fibrosis and tubular atrophy; OR, odds ratio.
predictor of long-term graft outcome. Hirt-Minkowski et al. confirmed in their study that urinary CCL2:Cr and CXCL10:Cr measured 6 months after KTx provide prognostic information regarding renal graft outcomes and they have similar performance. However, that study included 185 patients, of which 52 reached primary outcome at a median 6 years. 48 Our results could be explained by smaller number of patients in the study and shorter time of observation. Moreover, Raza et al., in the study involving 273 patients, proved that urinary CXCL10 is significantly elevated at the time of renal allograft rejection. 20 There are several limitations of the study. It is singlecenter, includes relatively small sample size, and it is based on previous study. Sample size limited the application of the multivariable model. Another limitation is that concentrations of urinary chemokines were measured only 1-year after transplant at the time of biopsy. One more concern is that all biopsy specimens were assessed by one pathologist. Moreover, mortality from all causes was considered in all patients, including patients with failed graft, in which dialysis could increase the risk of death.

| CONCLUSIONS
Chemokines are easily, noninvasively detected in urine by ELISA, which could facilitate their application in clinical practice. In the era of personalized medicine, urinary CCL2:Cr can be considered as a factor providing complementary information regarding the risk of graft failure or increased mortality.
It could influence individualized therapeutic interventions and follow-up strategies.